The present invention concerns dietary fat compositions in accordance with patent claims 1 and 2, and food products in accordance with patent claim 6.
The invention also concerns plant stanol derivatives in accordance with patent claim 15, plant sterol and/or plant stanol dicarboxylic acid derivatives in accordance with patent claim 21, amino acid derivatives in accordance with patent claim 22, citric acid derivatives in accordance with patent claim 23, tartaric acid derivatives in accordance with patent claim 24, and plant sterol and/or plant stanol 3 (R)hydroxybutyric acid esters and their derivatives or salts in accordance with patent claim 25.
The method here described also concerns a method to make phytostanol esters in accordance with patent claim 26.
Plant sterols or phytosterols refer to the sterols appearing in the plant kingdom which closely resemble cholesterol in terms of structure. They are, like cholesterol in mammals, a certain structural component of external and internal membranes and thus essential constituents for the living functions of cells. Isolated plant sterols often appear in poorly soluble, crystalline form. The phytosterols appearing in nature are intrinsically fat solutions, however. Chemically, natural sterols are C26 . . . C30 alcohols, which have an aliphatic side chain in the C-17 position.
Cholesterol (5-cholestene-3-xcex2-ol) and its hydrogenated form cholestanol ((3xcex2, 5xcex1) cholestane-3-ol) are found mainly in humans and animals. The sterols found in the animals, plants and mushrooms of marine organisms and sea-weeds form a wide variety of oxidation, double bond, methyl group substitution and C-17 side-group structures. The a configuration of the C-5 position when a hydrogen atom is linked to it is common to the natural sterols. A small number of plant stanols are also found in plants, the isolation of which is not economically profitable. Using a catalyst, phytosterols isolated in commercial applications can be hydrogenated into corresponding stanols.
Many phytosterols (independent of origin) are closely reminiscent of cholesterol in structure. The most well-known and the most studied are e.g. xcex2-sitosterol (24xcex2-ethyl-xcex945-cholestene (S24(xcex2)-ethyl-xcex945-cholestene-3xcex2-ol). The sterol that is characteristic of yeast and mushrooms is ergosterol (xcex224-methyl-22,23-dehydro-xcex945, xcex947-cholestadien-3xcex2-ol=5,7,22-ergostatrien-3xcex2-ol; provitamin D). The anticholesteric nature of the two first-mentioned phytosterols is generally known. It is possible that other sterols suited for this purpose can be found in nature, in addition to the xcex2-sitosterol.
Plant sterols form part of our natural nourishment. The sources of plant sterols in our diet include plant oils and the margarines made from them, while phytosterols can also be found in grain products, soy beans and rice. The regular daily diet includes 0.2-0.3 g of plant sterols.
A certain physiologically important group of compounds is formed by relatives of the sterols, the cholic acids whose role in the food digestion organs is to act as a xe2x80x9cbiological soapxe2x80x9d, as an emulsifying agent of fat and as an absorption aid. Human bile contains several cholic acids conjugated with glycine and taurine (2-aminoethanesulphonic acid); glycine conjugates are those which mostly appear.
The effect preventing the cholesterol absorption of the xcex2-sitosterol is assumed to be based on its ability to displace the cholesterol molecule in cholic acid/fat micelle. (Ikeda et al. 1989, J. Nutr. Sci. Vitaminol. 35:361-369). Many pieces of research into plant sterols have also shown that crystalline phytosterols do not dissolve very effectively in the micelle phase and therefore are not able to effectively prevent the absorption of cholesterol from the digestive tract. When water is present, solubility of phytosterols in plant oils is restricted to 2% at room temperature and to 3% at body temperature.
According to the observations of Heineman et al. 1991 (Eur. J. Clinic. Pharmacol. 40 Suppl. 1, p. 50-63), plant sterols inhibit cholesterol absorption only in fat-soluble form. A saturated form of xcex2-sitosterol, xcex2-sitostanol, inhibited cholesterol absorption in an infusion test substantially more effectively than the corresponding sterol (83% vs 50%). In general, the plant sterols are absorbed into the blood circulation poorly and the stanols not at all. Normally, the concentration of plant sterols in serum is {fraction (1/300)}th part of the serum cholesterol level. In addition, according to the observations of Miettinen et al. (U.S. Pat. No. 5,502,045), an increase in sitostanol concentration in nutrition also lowers the measured xcex2-sitosterol and campesterol concentration of the blood serum.
According to the observations of Mattson et al. (1977, J. Nutr. 107:1139-1146), it is probable that the molecules that really prevent cholesterol absorption are free phytosterol molecules which are hydrolytically released from their esters. This argument is supported by the fact that the cholesterol absorption lowering effect of the phytosterols does not depend on the length of the carbon chain of the fatty acid. Molar quantities of the fatty acid esters such as acetate, decanoate and oleate are equally as effective. In addition, the dicarboxylic acid esters such as phytosterol hemisuccinate lower the cholesterol content of blood as effectively as the phytosterol monocarboxyl acid esters.
The previously known methods to take advantage of the hydrophilic derivatives of xcex2-sitosterol in food are based on a particular compound""s glycosides obtainable from nature, e.g. the xcex2-sitosteryl-xcex2-D-glucoside. The carrier in the aqueous solution is, for example, lecithin, while the solvent may be isopropanol and the additive isopropylmyristate. In addition, the sterols"" glycoside derivatives are bonded from the alcohol solution and/or emulsion to the starch (DE publication 2113215).
GB patent publication 938 937 describes a synthetic chemical, water-soluble phytosterol hemisuccinate-polyethyleneglycol condensate which has proved, in animal tests, to be effective in lowering blood cholesterol concentrations. The publication does not, however, state an opinion on the compound""s hydrolytic decomposition in the system, or whether this kind of derivative is absorbed into the blood circulation or not. The compound is planned for use as a component of various juices, soups and soft drinks. It is clear, however, that the widespread use of the compound in food will be limited by the ethyleneglycol-based polyether component it contains and which is foreign to the human body.
Herting and Harris (1960, Fed. Proc. 19:18) have reported on a water-soluble soy sterol-2-carbamate glutaric acid K+xe2x80x94salt which has been proved to lower blood cholesterol concentrations. Carbamates can be made by e.g. a reaction between chloroformates and primary amines. The carbamine acids are unstable and break up, releasing amine and carbon dioxide. The salts of carbamine acids are much more stable, however. As the chemical environment of food varies greatly, one can justifiably assume that there will be significant restrictions to the use of the compound. The publication does not indicate whether the presented compound is planned at all for use in foods.
A rise in the blood cholesterol levels is among the more important risk factors, along with smoking and higher blood pressure, in the spread of cardiac and vascular diseases. U.S. Pat. No. 5,502,045 describes the production of xcex2-sitostanol fatty acid esters and their addition to food, especially as a part of edible fats in margarine, for example. The research carried out by Miettinen et al. 1995 (New Engl. J. Med. 333: 1308-1312) observed a decrease in the total serum cholesterol when the daily supply of plant sterol of the test individuals had stabilised at the 2-3 g level. The test used a saturated form of plant stanol, sitostanol, whose fat solubility had been increased by the esterification process. The tendency of the plant sterols to oxidate was reduced by the hydrogenation process.
A sitostane-3xcex2-, 5xcex1-, 6xcex2-triol-3,6-diformiate (SU 574-450) classified as a stanol derivative has also been made from xcex2-sitosterol. The compound has been proved to moderately lower the triglyceride content of the blood serum of rats and effectively lower the triglyceride content of liver (54% in guinea pigs). Tests measuring acute toxicity and performed on white male mice did not show toxicity at a 3.5 g dose per 1 kg of the animals live weight. The xcex2-sitosterol used as a source material did not give a positive response in this test. The compound is planned for use in the prevention or treatment of fatty liver. The compound is fat-soluble. It does not dissolve very well in alcohol and not at all in water.
In patent publication EP 0 430 078 concerning cholesterols and phytosterols, a large number of (alkylene-ester/ether/amide functional hydroxyphosphine) coline hydroxide salts were made, which can be taken in doses orally in the form of tablets, capsules, emulsions, suspensions and in soluble form in order to lower the cholesterol content of blood plasma. The patent publication mentions as a synthesis intermediate stage a group of sterol and stanol hydroxy acid derivatives, namely cholest-5-en-3xcex2-yl-(S)-2-hydroxypropionate, stigmasta-5.22-diene-3-xcex2-yl-3-hydroxypropionate, stigmastanol-3-xcex2-glycolate, stigmastanol-3-xcex2-hydroxypropionate and stigmastanol-3-xcex2-4-hydroxybutyrate (=xcex3-hydroxybutyrate). The aforementioned hydroxy acid esters were not used or tested, however, as nutritional additives to lower the cholesterol in blood. According to the patent publication, the synthesis of the compound group requires very powerful and toxic reagents such as phosgene, oxalyl chloride, trityl chloride or phosphoryoxy chloride, so that it can justifiably be assumed that their use is restricted to pharmaceutical preparations. The publication expresses no opinion on the behavior of sterol derivatives in the digestive system.
xcex2-sitosterols have been fused with HO2C (CH2)nCO2H (n=0-8) di-acids at a 40-50% supply and the liquid crystallization of the resulting compounds has been examined (Mukhina et al. 1977. Leningr. Khim-Farm. Inst., Leningrad USSR 24. Obshch. Khim. 47(6):1429-1430). The liquid crystals have been conventional applications of sterols e.g. cholesterol esters. No reports have been made, however, on the application of these compounds in lipid chemistry and the manufacturing process of food products.
Previously known are the 2-(2xe2x80x2-alkenyl) succinates made from cholesterols and phytosterols (EP 0554 897) and their use in cosmetics. The use of the C2 . . . C6 hydroxy acid esters of phytosterols as components of hair shampoo compositions is known from patent publication JP 09194345. Phytosterols have also been esterified with lactic acid-oligomers and the resulting sterol esters have been used in formulations usable in e.g. hand creams and lip sticks (JP 58008098).
The use of phytosterols and their natural metabolites such as deoxycoline acid ascorbic, glutaric, tartaric and lactic acid esters for medical purposes other than the prevention or treatment of hypercholesterolemia is known from patent publication DE 19701264.
The xcex2-sitosterol has also been esterified with 2,3-dihydroxy cinnamic acid and the antioxydative properties of the compound have been studied (Takagi, T. and Iida, T. J Am Oil Chem Soc 57(10):326-330 (1980).
Citric acid has previously been used to make fat-soluble glyceride derivatives, which observably have been effective antioxidants in plant oil and food which has a high fat content (Qiu et al. Zhogguo Youzhi. 1996,21,3,14-18). In addition, a synergetic antioxydative effect with tocopherol has been noticed in the case of citric acid, monoacyl glycerol citrate and ascorbic acid (Aoyma et al., Yakagaku, 1985.34(1), 48-52.
In previously known methods a phytosterol or phytostanol derived from soy oil is dissolved in some edible medium (U.S. Pat. No. 5244887), which is used as a suspension or dispersion in food products. Generally known dispersing agents such as lecithin are used as additives.
Previously known techniques also include methods, in which hydrophobic sterols and stanols and their fatty acid esters are dissolved or fixed in e.g. fat and phospholipid emulsions (DE publication 4038385).
According to the the prior art, a fat-soluble xcex2-sitosterol derivative is usually made from methyl esters by transesterification. Other generally known esterification methods are the reaction of sterols with fatty acid chlorides and anhydrides. Stanols, which are hydrogenated forms of plant sterols, can be used in the manufacture of esters.
The natural tendency of sterols to oxidize is reduced chemically by the hydrogenation process. In addition, the stereochemical structure of the molecule is altered by the hydrogenation process. In catalytic hydrogenation, the main product is the trans-fusion of the A, B rings, i.e. the hydrogen in the C-5 position is xcex1-orientated, so that the molecule is almost flat. A small amount of C-5 position xcex2-orientated hydrogen (cis-fusion) is obtained as a byproduct, whereafter the molecule has a curved shape. Trans-fusion is characteristic for natural hormones and cis-fusion for cholic acid.
According to the prior art, heterocyclic derivatives of xcex2-sitosterol are manufactured by making the hemisuccinate of the xcex2-sitosterol react with SOCI2 and thiols, amines and phenols. The antilipemic properties of the esters obtained have been investigated, but there are no reports of the interaction between the ethanol/lipid soluble hemisuccinate of xcex2-sitosterol with water soluble compounds (Arch. Pharm. (Weinheim. Ger.) (1990), 3232(7), 401-4).
The aim of the present invention is to synthesize new phytosterol and phytostanol esters, preferably xcex2-sitosterol and xcex2-sitostanol esters that have been modified so that the fat solubility of their derivatives has significantly increased in relation to free phytosterols and phytostanols. The aim of the invention is particularly to create tailor-made functional derivatives from phytosterols and phytostanols, that when dissolved in lipids can inhibit the absorption of cholesterol, and also increase the interaction between the hydrophobic lipid phase and water phase.
One risk factor in daily dietary intake is also the salt used in preservation, and regulation of taste and structural properties of industrially prepared food products, such as margarine, mayonnaise, and spreads, of which consumption (in excess quantities) is believed to be a significant factor in the development of hypertension.
Other commonly added ingredients to fat-containing food products include emulsifiers, thickeners, antioxidants, acidity regulators, and aromatic supplements. Food products may also contain colors, such as xcex2-carotene, and vitamins, such as vitamin A and D.
The aim of the present invention is also to improve the structure, taste and preservation of dietary fat products.
The invention is based on the ability of the HLB relationship (Hydrophilic Lipophilic Balance) of phytosterol or phytostanol, preferably xcex2-sitosterol or xcex2-sitostanol esters, to be regulated so that the compound is soluble in lipid and ethanol, and dispersible in water. The invention is also based on the fact that attaching a chemically very different group to a phytosterol or phytostanol molecule decreases the characteristic crystallization of sterols and stanols, e.g. in a plant oil solution. According to the invention, xcex2-sitosterols or xcex2-sitostanols can be made to react with aliphatic hydroxy acids, ketoacids, dicarboxylic acids or amino acids, or their derivatives, to form xcex2-sitosterol or xcex2-sitostanol esters with the aforementioned acids. The esters formed are lipid soluble, and improve the surface chemical mixability of fat and water.
The compounds according to the invention have a highly lipophilic sterol/stanol structure and a hydrophilic structural unit.
More precisely, it is characteristic for the phytosterols and phytostanols according to the invention that the hydroxyl group in the 3xcex2-position in the sterol/stanol ring is esterified with a polar substituent, for example a dicarboxylic acid, hydroxy acid, amino acid, or with the oligomeric polyesters of the aforementioned acids or with mixed esters of the aforementioned dicarboxylic acids or hydroxy acids, with either an alcohol component or fatty acid component.
Significant advantages are achieved with the invention. Polar, lipid-soluble phytosterol and phytostanol derivatives according to the invention can be utilized to emulsify lipids in water; in other words, the compounds work using a mechanism that is almost the same as the body""s own lipid emulsifying mechanism. The present invention is based on the observation according to which, for example phytosterol dicarboxylic acid hemiester, plant oil, and water form a stable emulsion when mixed. Thereby, compounds according to the invention are excellent for application in the preparation of e.g. light spreads, mayonnaise, and salad dressings.
Compounds according to the invention may be added to products that typically contain plant oil, such as cooking oil, margarine, light spreads, mayonnaise, chocolate, and ice cream, in comparative concentrations (preferably 0.1-10% of total fat mass), in comparison to already commercialized phytostanol fatty acid ester-containing dietary fat preparations. Products according to the invention can also be added to industrially prepared fat emulsions, such as cream liqueur bases.
More precisely, dietary fat constituents according to the invention are those constituents defined in the characterizing part of patent claims 1 and 2, and the food product defined in the characterizing part of patent claim 6.
The invention differs significantly from previously known solutions in which sitostanol, which lowers blood cholesterol levels, is present as a fatty acid ester in one component of dietary fat. The energy content of the dietary fats containing compounds according to the invention is significantly lower (5-10%) than the sitostanol fatty acid esters.
The phytosterol and phytostanol dicarboxylic acid and hydroxy acid esters according to the present invention are hydrolyzing esters, whose characteristics can be assumed to differ from those stanol esters that are already known and have been commercialized. Factors affecting the different rates of hydrolysis of different esters are known from general organic chemistry. Steric and electronic factors are the most important. A substituent group in the side chain of an ester that attracts electrons, for example, increases the speed of hydrolysis. On the other hand, a substituent, such as an alkyl group, that donates electron density in the side chain decreases the speed of hydrolysis. Therefore, for example, poly(hydroxy succinate) is hydrolyzed faster than poly(2-S-hydroxy propionate).
Plant sterol and/or plant stanol esters according to the invention can be added, for example, to food products containing plant oil, such as mustards, salad dressings, and peanut butter. Citric and/or tartaric acid, or their salts, are often added to the aforementioned food products. Phytosterol citrates, lactates or tartrates according to the invention can also be used to achieve the same effect in these food products. The unhealthy effects of a meal that is high in fat and cholesterol can be counteracted with the use of these products.
Dissolved in plant oil, the plant sterol and/or plant stanol derivatives according to the invention can also very well be added to e.g. industrially produced canned fish, of which two common examples are tuna or sardines preserved in plant oil.
Of the compounds according to the invention, phytosterol and phytostanol lactic acid esters (-mono-L-lactate and -poly[L-lactic acid]) are lipid soluble compounds that have a potentially wide range of application, and whose range of application ranges from margarine to various milk products. As the compounds according to the invention have been found to dissolve and be excellently compatible with various fat dispersions, there is no reason not to add the compound in question, preferably lactic acid esters, to e.g. cream, which can then be used to make ordinary processed products, such as soured whole milk and sour cream.
In terms of its physical properties, xcex2-sitosterol and xcex2-sitostanol-oligo-/poly-L-lactic acid ester is a viscose liquid between 50-100xc2x0 C., which can be dispersed in water with common methods, if necessary. If desired, the regular emulsifiers used in food products, such as sugar lipids and lecithin, can be used as additives, and proteins, modified starches, hydrolyzed carboxy methyl cellulose are some examples of substances that can be used as protective colloids.
Compounds according to the invention are soluble and/or dispersable in water, ethanol and lipids, so that compounds according to the invention can at the same time be dissolved/dispersed in water and ethanol and dissolved in the fatty component of the product.
Compounds according to the invention can be e.g. in an approx. 6% fat-containing cream liqueur dissolved simultaneously both in a water/alcohol solution and in the fatty component of the product.
Compounds according to the invention dissolve and/or disperse in water, ethanol and lipids, and also acetic acid, so that compounds according to the invention can at the same time be dissolved/dispersed in water and acetic acid and dissolved in the lipid component of the product.
Chemically, plant sterol and plant stanol esters according to the invention can be classified as hydrolyzing esters. Even though we are not able to commit to any theory regarding the action mechanism of the compounds, we can justifiably assume that the compounds according to the invention act as substances that lower the serum cholesterol level, and that, in the body, the carboxylic acids act in accordance with their characteristics.
The sterol or stanol derivatives according to the invention is based on natural compounds, whose use in food products as safe techno chemicals and as materials suitable for a biological environment is anyway widespread. In addition, they can be synthesized economically and with a minimum number of side products. Here, processes producing side products mean, for example, the acid chloride method, in which 1 mole of sterol reactant produces 1 mole of hydrochloric acid, and the anhydride method in which the mole amount of reactant produces the same number of moles of carboxylic acid, or the reaction of a sterol with an enol ester (e.g. propene acetate) which produces the same number of moles of acetone.
Compounds according to the invention, e.g. citrate and tartrate, are examples of a group of organic esters which are known for being difficult to absorb. In large doses, these organic ions have been used as osmotic laxatives. (Tuomisto, J. and Paasonen, M. 1982. Pharmacology and Toxicology (in Finnish). Kandidaattikustannus Oy. Helsinki. pp. 526-529).
The invention will now be explained in more detail with the aid of the following detailed description and with the reference to a number of working examples.
The aim of the invention here presented is to produce a polar ester from xcex2-sitosterol or xcex2-sitostanol that is soluble in dietary fats, and which has a carboxylic acid component that is a natural compound found in the body and/or is found and/or is useable in food products, preferably succinic acid, glutaric acid, ketoglutaric acid, malic acid, tartaric acid, citric acid, lactic acid, 3(R)-hydroxybutyric acid or an amino acid derivable from proteins, or a derivative of these.
The hydroxy acid ester of a phytosterol and phytostanol can also be an oligomeric polyester, that is defined by the more general definition polyhydroxy alkanoate. In this case, poly(L-lactic acid) is also classified as a polyhydroxy alkanoate.
Hydroxy acid esters of phytosterols, such as citrate and tartrate, can be modified if desired by e.g. mixed esterification, when either the COOH-group is esterified using an aliphatic alcohol, polyol, polyol (C2 . . . C22) fatty acid ester, or a free OH-group is esterified with C2 . . . C22 fatty acids.
If required, hemi esters of phytosterol and phytostanol can be modified by mixed esterification with aliphatic alcohol, polyol, or with a polyol(C2 . . . C22) fatty acid ester.
The most preferred polyol component is glycerol, though it can also be ascorbic acid.
Generally speaking, it is characteristic for the invention that the polarity of the xcex2-sitosterol/stanol esters is increased by increasing the number of heteroatoms, such as N and O in the molecule. Even more generally, it is characteristic for the invention that the substituent in the C3 position of the phytosterol/stanol decreases the crystallization of the derivative, compared to an unsubstituted plant sterol or plant stanol, and thus increases the lipid solubility of the compounds in question, or the stability of the fat solutions. Suitable functions in the side group include ester, ether, carbonyl, carboxyl, amine and amide and/or e.g. combinations of these functional groups.
According to certain preferred embodiments in the present invention, phytosterols and phytostanols are specifically esterified with lactic acid, citric acid, succinic acid or glutaric acid.
Particularly preferred compounds according to this invention include the esters formed by plant stanol with the acids listed above, and their derivatives.
Particularly preferred compounds for addition to dietary fat products are the dicarboxylic mixed esters of plant sterols and plant stanols according to this invention, such as ethyl xcex2-sitosterol succinate, xcex2-sitosterol or xcex2-sitostanol lactate, xcex2-sitosterol or xcex2-sitostanol succinate lactate, xcex2-sitosterol or xcex2-sitostanol hydroxy butyrate, xcex2-sitosterol or xcex2-sitostanol tartrate, xcex2-sitosterol or xcex2-sitostanol succinyl 3(R)-hydroxy butyrate and acylated, mix esterified citrates.
Dicarboxylic acid derivatives of plant sterol or plant stanol have the formula:
Sxe2x80x94O2CRCO2Yxe2x80x83xe2x80x83(I)
in which
S=a plant sterol or plant stanol
R=C2 . . . C6 carbon chain;
Y=H or C2H5 
In this invention, preferred compounds are, for example, sterol succinate lactic acid condensate (II), sterol succinate-3(R)-hydroxy alkanoate condensates (III), such as sterol succinate-3(R)-hydroxy butyric acid condensate (IV)
Y=xe2x80x94CH(CH3)(CO2Y1)*(2S)xe2x80x83xe2x80x83(II)
Y1=H or C2H5 
or
Y=CHCH2CO2Y2I(*)R1xe2x80x83xe2x80x83(III)
Y2=H or C2H5,
R1=C2 . . . C6 alkyl
or
Y=CHCH2CO2Y3I(*)(3R)CH3xe2x80x83xe2x80x83(IV)
Y3=H or C2H5 
According to this invention, amino acid esters of phytosterols or phytostanols are also preferred compounds, and have the formula: 
S=plant sterol or plant stanol
R=H or the structure appearing in natural L-amino acids
According to this invention, citric acid derivatives of phytosterols or phytostanols, are also preferred compounds, and have the formula: 
S=plant sterol or plant stanol
R1=R2=H and R3=C2 . . . C22 carboxyl acid residue
or
R1=R2=xe2x80x94C2H5 and R3=H
or
R1=R2=xe2x80x94C2H5 and R3=C2 . . . C22 carboxyl acid residue
or
R1=R2=xe2x80x94CH2CHOHCH2OH and R3=H
or
R1=R2=xe2x80x94CH2CHOHCH2OH and R3=OCCH3 
Therefore, preferred citric acid esters are, for example, steryl acyl citrate, steryl diethyl citrate (R1=R2=C2H5, R3=H), steryl acyl diethyl citrate (R1=R2=C2H5, R3=C2 . . . C22 carboxylic acid), steryl citrate diglycerol ester (R3=H, R1=R2=xe2x80x94CH2CHOHCH2OH), steryl acetyl citrate glycerol ester (R3=OCCH3).
According to this invention, tartaric acid derivatives of phytosterols or phytostanols, are also preferred compounds, and have the formula:
Sxe2x80x94O2CCH(OR1)CH(OR2)CO2Yxe2x80x83xe2x80x83(VII)
S=plant sterol or plant stanol
R1=R2=Y=H;
or
R1=R2=H and Y=C2H5 
or
R1=R2=acyl C2 . . . C22 and Y=H
or
R1=R2=acyl C2 . . . C22 and Y=C2H5 
The aim of the invention is to provide a method for the use of tailor-made functional plant sterol and pant stanol esters as a cholesterol-lowering component in dietary fats and fat-containing milk products or alcoholic beverages. In addition, the aim of the invention is to improve the quality, preservability, and processability of these products.
xcex2-sitosterol and/or xcex2-sitostanol esters can be dissolved in animal fats, plant oils or 4-40% fat emulsions, in which the fat proportion preferably contains 1-10 weight-% of compounds according to the invention. Alternatively, liquefied xcex2-sitosterol or xcex2-sitostanol esters (preferably lactic acid esters) can be dispersed in water (preferably to form a 10-40% emulsion), and added in the required concentration to the food product being prepared.
xe2x80x9cPlant sterol and/or plant stanolxe2x80x9d refers to sitosterol, stigmasterol, campesterol, brassicasterol, cycloartenol, 24-methylene cycloartenol and cyclobranol and/or their hydrated forms, and mixtures, and which have a serum cholesterol lowering effect. Even though the terms xc3xa2-sitosterol and/or xcex2-sitostanol are used in conjunction with this invention, it also includes other plant sterols and/or stanols which have a cholesterol-lowering effect.
By a xe2x80x9cdicarboxylic acidxe2x80x9d here is meant an organic acid that has two carboxyl groups (xe2x80x94COOH). Suitable dicarboxylic acids in this invention are, for example, glutaric acid and its derivatives, and succinic acid.
In an esterification reaction, the xe2x80x94OH group of the carboxylic acid is released and forms water, while the alcohol, in this case xcex2-sitosterol or xcex2-sitostanol, reacts with the carboxylic acid residue to form an ester.
By a xe2x80x9chemi esterxe2x80x9d here is meant that only one of the dicarboxylic acid""s xe2x80x94COOH groups has reacted with the alcohol.
By a xe2x80x9cketoacidxe2x80x9d here is meant an organic acid that contains a keto-group in the hydrocarbon structure of the acid. Ketoglutaric acid is an example of a ketoacid.
By a xe2x80x9chydroxy acidxe2x80x9d here is meant an organic acid that contains a hydroxyl group in the hydrocarbon structure of the acid. In this invention, suitable hydroxy acids include malic acid, tartaric acid, citric acid, lactic acid, and 3(R)-hydroxy butyric acid.
By an xe2x80x9camino acid ester of xcex2-sitosterolxe2x80x9d is meant, for example xcex2-sitosterolglycinate (=xcex2-sitosterol amino acetic acid ester). The amino group may, of course, have originated from any amino acid, poly(amino acid) or peptide.
xe2x80x9cPhytosterol/stanol polyhydroxy alkanoatesxe2x80x9d refers to the following, common chemical structures: 
A xe2x80x9cxcex2-sitosterol oligo/polyesterxe2x80x9d refers to the formation of a xcex2-sitosterol ester from more than one hydroxy acids or with a dicarboxyic acid or amino acid. One example of such an ester is the xcex2-sitosterol succinate (acyl glycerol) ester.
xe2x80x9cPolyolxe2x80x9d refers to a compound containing at least two hydroxyl groups. Glycerol is an example of a polyol.
By xe2x80x9cpoly/oligo esters of xcex2-sitosterol or xcex2-sitostanolxe2x80x9d is meant that xcex2-sitosterol has been esterified with, for example lactic acid polymer which has at least two L-lactic acid units.
By xe2x80x9cdietary fatsxe2x80x9d is meant animal or milk fats adapted for use as food, plant oils, such as rape seed, sunflower, olive and palm oil, either in their natural form, or chemically modified.
According to this invention, the fat proportion of a food product can contain 0.01-10 weight-% of phytosterol/stanol, preferably the hydroxy acid, dicarboxylic acid, or amino acid derivatives of xcex2-sitosterol or xcex2-sitostanol, or mixtures of the aforementioned derivatives.
Fat-containing food products refers to such food products of which one component is fat derived from either animal or plant sources. Such food products are, for example, chocolate, cream liqueur, ice cream, butter, margarine, sandwich spreads, mayonnaise, salads containing mayonnaise, salad dressings, mustards, and preservatives, as well as various milk products. Food products containing fat also includes fat-containing alcoholic beverages.
It is characteristic for the method according to the invention that xcex2-sitosterol or xcex2-sitostanol is esterified in a one-step process with a dicarboxylic acid, hydroxy acid or amino acid, so that there will be at least one carboxylate, hydroxyl, or amino group, or a combination of the aforementioned functional groups in the ester""s side chain. Also characteristic of the method is that the sterol/stanol esters are soluble in fats or fat emulsions, in which form they can be used as a structural component of food products prepared from fats or fat emulsions.
In principle, the amino acid esters of xcex2-sitosterol or xcex2-sitostanol can be made using any generally known peptide-synthetic method, for example, the activated esters (p-nitrophenyl, hyrdoxy succinic imide, pentachloro phenyl esters) of amino acids together with an N-protection (for example, BOC=tert-butyl oxycarbonyl and benzyl oxycarbonyl). It is preferred to use benzyl protection, because as a powerful protective group it permits, for example, transesterification when making sterol esters. Benzyl protection is particularly preferred for a secondary amino group. L-proline and 4-hydroxy-L-proline are examples of such groups. In addition, benzyl protection can be removed by hydrogenation.
Compounds according to the invention are manufactured by combining the reactants in the desired molar ratios (batch process) and removing water (preferably azeotropically or by molecular sieving), alcohol, or other volatile condensation products from the reaction mixture by distillation, giving a high yield of esters that are soluble in lipids or ethanol.
The esterification of a xcex2-sitosterol or xcex2-sitostanol is carried out at a high temperature 110-280xc2x0 C., either by a direct esterification method, in which water is removed azeotropically from the reaction mixture, or by transesterification, when some volatile condensation product is distilled from the reaction mixture. Hemiesters (for example, succinate and glutarate) are more economically manufactured directly from the corresponding cyclic anhydride at a temperature of 90-200xc2x0 C.
In esterification based on the removal of water, it is technically possible to use numerous azeotrope systems, advantageously hydrocarbons, such as cyclohexane, toluene or technical xylene. The task of the aforementioned solvent in the reaction mixture is not only water removal, but also the control of the temperature of the reaction mixture. More precisely, the temperature of the reaction mixture is controlled by adjusting the ratio of the weights of the solvent and the reactants to 15-50% of the mass of the reactants.
In esterification at a temperature of less than 140xc2x0 C., it is preferred to use cyclohexane or toluene as the solvent and xylene at higher temperatures. Technically, it is also preferred to allow water, which is possibly in the reactants, to leave as an azeotrope, before arriving at the reaction temperature; thus a separate drying stage for the reactants may be unnecessary.
In direct esterification, it is also preferred to use a strong acid as the catalyst, which need not be removed from the reaction mixture. Esterification with cyclic anhydrides can also be carried out thermally without a catalyst. Esterification can be carried out with cyclic esters, such as lactons, glycolides and lactides, using the conventional methods known to the literature of chemistry.
According to one preferred embodiment, phytosterol/stanol oligomeric lactates are manufactured from compounds according to the invention by non-catalytic esterification at a temperature of 155-120xc2x0 C., starting from a 90% L-lactic acid water-based solution and phytosterols or phytostanols, with toluene acting to form a water azeotrope. Under such weak reaction conditions, it is justified to assume that the risk of racemization of the lactic acid is very small.
The catalyst used in transesterification can be equally well strong acids (TsOH (=paratoluene sulphonic acid), H2SO4, H3PO4, etc.), alkalis (NaOH, KOH, NaOEt, t-BuOK (=potassium tert-butylate) etc.) or the generally used weak Lewis acids. From the point of view of the intended application of the present invention, it is not, however, preferred for metals to appear in the reaction mixture, except for Na, K, Ca, Mg. Generally, it is not preferred from the point of view of the intended application for halogenides to appear in the reaction mixture.
According to one preferred embodiment of the invention, xcex2-sitosterols are generally hydrogenated using known methods to form respective stanols, and the esterification reactions are only carried out after this using stanols.
According to instructions in literature, (Fieser and Fieser, 1967. Reagents for Organic Synthesis, Vol. 1. p. 799) sterols can be hydrogenated, at a high yield and in moderate reaction conditions, to form stanols (0.02% PtO2 of the mass of the sterol. EtOAc, H2/103kPas, 40-5xc2x0 C., xc2xd h, acid co-catalyst). The reaction products are 88% sitostane-3xcex2-ol, 3.4% sitostane-3xcex1-ol and 0.9% sitostane. In addition to this, 1.6% sitostane-3xcex2-acetate is obtained from the exchange reaction.
According to one preferred embodiment of the invention, xcex2-sitosterols are esterified and sterol esters are hydrogenated to form stanols. Because, for example sterol lactates are highly soluble in alcohol, a sterol ester can be hydrogenated to directly form its respective stanol ester. This achieves the significant advantage that hydrogenation can be carried out at room temperature. As the solubility of phytosterol in alcohol at 20xc2x0 C. is xe2x89xa61%, phytosterols cannot be hydrogenated at similar conditions to form stanols. Sterols must be hydrogenated to form stanols in ethanol at 65-77xc2x0 C.
Sterol esters that are soluble in acetic acid can be hydrogenated in concentrated acetic acid to form stanols. It is practical for the hydrogenation to be carried out directly in an ethanol or acetic acid solution, which will be used later in the manufacture of beverages or vinegar products.
A large number of heterogenic and homogenic hydrogenation catalysts and reagents are known in organic chemistry (e.g. March, J. advanced Organic Chemistry, Reactions, Mechanism and Structure, 4th edition. Chapters 5-9 and in the notes to them), by means of which the double bond of a sterol can be reduced without converting the functional group of the 3xcex2-substituent, which can be, for example OH, COOH, CONHR or COOR.
The polarity of the sterol/stanol esters described above is an extremely useful property. It is characteristic of compounds according to the invention, e.g. xcex2-sitosterol dicarboxylic acid hemiesters and citrates that in lipid solutions (e.g. 2.5-5% of the fat component) they promote the mixing of the lipid and the water. This was found to have a preferred effect on, e.g. the structure and taste of light spreads, which in this case means that the product is less oily; excellent to spread, at both room and refrigerated temperatures. The prepared light spread also lasted better than the comparative sample when heated to 45xc2x0 C. for up to 4 hours. By improved thermal resistance is meant that after 2 hours at 45xc2x0 C., more oil had separated from the comparative sample than from the sterol-containing sample. Over the next 2 hours, the separation of oil from the comparative sample continued. In the sterol-containing spread, however, negative change had ceased. Changes in the sterol ester-containing sample, in which the fat component contained 5% plant sterol hemisuccinate, were almost reversible; in other words, the structure of the sample returned to almost the same upon mixing the sample after it had returned to room temperature.
Generally, a 2.5-10% proportion of compounds according to the invention, e.g. sterol lactates, citrates, and hemisuccinates, in the fat component of the mass of dietary fat products, such as light spreads and mayonnaise, have been found to have no negative effects. Instead, it was surprisingly found that in most cases added salt can be left out of the product family in question, without the smell, taste or structure of the product being adversely affected in any way. In addition, sensory evaluations showed that sterol citrates emphasize e.g. the wine vinegar and mustard aromas contained in mayonnaise.
Just as with dietary fat products, the compounds according to the invention were found to be suitable for chocolate products. The fatty component of these products can contain compounds according to the invention e.g. 2-5% without any adverse effect on the product""s structure or taste.
For an alcoholic beverage, which in this case means a cream liqueur, the results were consistent with the previously described products. The fatty component of the product can contain, for example 2-4 weight-% of compounds according to the invention without changing the characteristic smell or taste of the product.
A significant advantage for compounds according to the invention, and their use as a component in various daily dietary fat preparations, chocolate confectionery, or alcoholic beverages, is that phytosterol/stanol hydroxy acid esters are potential antioxidants on the basis of what is already generally known e.g. about esterified citrates.
The following examples illustrate the invention. They do not, however, limit the patent""s scope of protection.
The starting material for compounds according to the invention was a plant sterol mixture (Weinstein Nutritional Products), which was composed of 45-55% xcex2-sitosterol, 20-30% campesterol, and 15-25% stigmasterol.